The invention pertains to the fields of fluid handling and electroosmosis. More particularly, the invention pertains to electroosmotic flow controllers.
Flow controllers are used to manage the flow of fluids through conduits. Traditionally, control of fluid volume and or fluid composition by flow controllers is accomplished by combinations of pumps and valves. In some applications, flow controllers are used to control fluid flows on the order of many milliliters or more per minute, while in other applications, the fluid flow rates are orders of magnitude smaller. Prior art flow controllers tend to suffer from a variety of shortcomings. In high flow and in low flow applications, prior art flow controllers have difficulty in maintaining precise flow rates in the face of changing head pressures, such as those generated by pumping devices. In addition, mechanical feedback loops used to control flow rates through flow controllers often introduce additional imprecision and dead space into flow-controlled devices.
Microfluidic systems are playing an increasingly important role in developing advanced separation techniques needed for chemical analysis. Currently, chemical separations and purification represent severe bottlenecks in many biotechnology and drug discovery applications. Traditional scale separation systems are not compatible with the speed and small sample sizes typically required for high throughput analytical techniques. Analogous to transistors on a computer chip, microfluidic separation systems can be easily fabricated and run in multiple parallel arrays. The increased throughput and speed attained will be critical to allow separation technology to keep pace with the explosive growth of biotechnological advancement.
The recently completed sequencing of the human genome has tremendously increased our knowledge of the role genes play in disease. Translating this knowledge into improved medical diagnostics and treatments requires a much more thorough understanding of biological processes at the cellular and molecular levels. One promising approach for developing the necessary understanding involves analyzing the expression of cellular proteins in normal and disease states. Another approach involves generating protein-protein interaction maps that characterize binding interactions among different proteins to better understand the diverse biochemical signaling pathways underlying normal and pathological cell physiology.
These analytical tasks are in many respects more challenging than genome sequencing efforts. In contrast to DNA, which is chemically and structurally similar irrespective of nucleic acid sequence, proteins have enormous chemical diversity. Thus, different proteins may have different molecular weights, different electronic charges, different solubilities, etc. depending upon their amino acid composition and post-translational modifications.
Therefore, the reliable microseparation techniques developed for gene sequencing are inadequate for protein analysis. Moreover, the genome is a static entity. It does not significantly change over time and identical sequences of DNA are found in almost all of the cells in the body. In contrast, the complement of expressed proteins (i.e., the “proteome”) in, for example, a red blood cell, is very different than that expressed, e.g., in a neuron, or a skin cell, or a liver cell. Also, the proteome changes as a function of age and disease state. Further, considering alternative splicing possibilities, it has been estimated that there are 100 times more proteins present than the number of genes. Given this formidable task, it is clear that fast, reproducible and robust separation methods are needed to thoroughly characterize protein expression levels and interactions.
Currently, cell protein extracts are most often analyzed using 2 dimensional gel (“2D gel”) electrophoretic techniques, followed by one or more rounds of mass spectrometry (“MS”). Many problems exist with 2D gels, however. Inter- and intra-laboratory reproducibility is notoriously poor. The analysis is time consuming, the 2D separation typically taking 1-2 days. After this, proteins on the gel must be stained, identified, cut out, extracted from the gel matrix, destained and serially loaded into a mass spectrometer. A complete analysis for a single cell can take months or longer.
An alternative analytical approach involves the use of gradient liquid chromatography (“LC”) followed by MS. While this technique does not have the separation capacity of 2D gel electrophoresis, it is faster and more reproducible. The proteins found in the effluent of the separation column can be fed directly into a mass spectrometer. Commercially available high performance liquid chromatography (“HPLC”) systems for carrying out analytical separations often use separation columns having diameters on the order of 2-5 millimeters and run at flow rates of a few milliliters per minute (“ml/min”). Since the flow rate into the MS is on the scale of microliters per minute (“μl/min”) or less, most of the effluent from the HPLC, including that containing possibly valuable sample, is thrown away. Using a typical HPLC unit, these tandem liquid chromatography/mass spectroscopy (“LC/MS”) analyses take about an hour.
Increasing the throughput of HPLC separations can be accomplished in several ways. For example, separations can be carried out more quickly and multiple systems can be run in parallel. Given the high cost and large size of conventional HPLC systems, it is impractical to run more than a few systems in parallel. Also, the speed at which a separation can be done is limited by the large sample requirements of the system.
Microscale HPLC systems can, in theory, address both of these needs. Moreover, microscale systems offer other advantages such as reduced waste generation and low sample volume requirements. However, most currently available micro HPLC systems are simply large scale systems outfitted with flow reducers and dampers, and as such are not truly microscale flow devices. The macroscale high pressure pumps used in these systems are often unable to meter fluid flow with sufficient accuracy to generate reliable and rapid gradients, which are critical to reproducible HPLC analysis.
The present invention addresses these and other shortcomings of the prior art by providing electroosmotic flow controllers capable of providing precise flow rates in high and low flow applications using a combination of pressure-driven and electroosmotically-driven flows.